Immunolocalization of LeAGP-1, a modular arabinogalactan-protein, reveals its developmentally regulated expression in tomato

 Arabinogalactan-proteins (AGPs) are highly glycosylated cell surface proteins that are thought to function in plant growth and development. The developmentally regulated expression of LeAGP-1, a novel and major AGP in tomato, was examined in different organs and tissues of tomato (Lycopersicon esculentum Mill. cv. UC82B) plants with an anti-peptide antibody (i.e. the PAP antibody) directed specifically against the lysine-rich subdomain of the LeAGP-1 core protein. During cell differentiation in tomato plants, LeAGP-1 was associated with cell wall thickening and lignification of particular cell types. Specifically, LeAGP-1 was detected in secondary wall thickenings of maturing metaxylem and secondary xylem tracheary elements in roots and stems, and in thickened cell walls of phloem sieve elements. However, LeAGP-1 was also present in thin-walled, cortical parenchyma cells of seedling roots as well as thick-walled collenchyma cells in young stems, both of which are not lignified. Based on these observed patterns, possible roles for LeAGP-1 in plant growth and development are discussed. Received: 17 August 1999 / Accepted: 7 October 1999     

Differential expression of four genes encoding 1-aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding

1-Aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14) is the key regulatory enzyme of the ethylene biosynthetic pathway and is encoded by a multigene family in Arabidopsis thaliana, tomato, mung bean and other plants. Southern blot analysis revealed the existence of at least five ACS genes in white lupin (Lupinus albus L.) genome. Four complete and one partial sequences representing different ACS genes were cloned from the lupin genomic library. The levels of expression of two of the genes, LA-ACS1 and LA-ACS3, were found to increase after hypocotyl wounding. Apparently, these two genes were up-regulated by exogenous IAA treatment of seedlings. The LA-ACS3 mRNA levels were also elevated in the apical part of hypocotyl, which is reported to contain a high endogenous auxin concentration. This gene may be involved in the auxin- and ethylene-controlled apical hook formation. The expression of the LA-ACS4 gene was found to be almost undetectable. This gene may represent a “silent” twin of LA-ACS5 as these two genes share a considerable level of homology in coding and non-coding regions. The LA-ACS5 mRNA is strongly up-regulated in the embryonic axis of germinating seeds at the time of radicle emergence, and was also found in roots and hypocotyls of lupin seedlings. Received: 19 July 1999 / Accepted: 3 March 2000     

The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds

 A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants). Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related (PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against fungal infection, though our results do not exclude other functions for this protein. Received: 15 March 1999 / Accepted: 12 July 1999     

Transgene expression driven by heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase small-subunit gene promoters in the vegetative tissues of apple (Malus pumila Mill.)

It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max [h.] Mers.), to drive expression of the β-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approximately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple. Received: 15 June 1999 / Accepted: 12 August 1999     

Molecular cloning and characterization of NFBP6, a putative floral homeotic gene, from tobacco

 NFBP6 cloned from tobacco is most closely related to petunia FBP6, a putative C-function gene. Also, NFBP6 is expressed specifically in stamens and carpels. Thus, NFBP6 provides a useful tool for molecular characterization of flower development.     

The genes ABI1 and ABI2 are involved in abscisic acid- and drought-inducible expression of the Daucus carota L. Dc3 promoter in guard cells of transgenic Arabidopsis thaliana (L.) Heynh.

 The ABA INSENSITIVE1 (ABI1) and ABI2 genes encode homologous type-2C protein phosphatases with redundant yet distinct functions in abscisic acid (ABA) responses. Results from Northern blot analysis showed that ABA- and mannitol-inducible expression of the COR47 and COR78/LTI78/RD29A (COR78) genes was more impaired in the abi2 mutant of Arabidopsis thaliana (L.) Heynh than in the abi1 mutant. Furthermore, ABA-plus-mannitol treatments were additive towards COR47 gene expression; however, the ABA-deficient aba1 mutant showed reduced COR expression relative to the wild type in response to mannitol and ABA-plus-mannitol treatments. These results support the notion that drought- and ABA-signalling pathways are separate yet overlapping. To facilitate quantitative analysis of the genetic control of tissue-specific ABA- and desiccation-response pathways, we analyzed ABA- and mannitol-inducible expression of a carrot (Daucus carota L.) Dc3 promoter:uidA (β-glucuronidase; GUS) chimaeric reporter (Dc3-GUS) in transgenic wild-type, ABA-deficient aba1, and ABA-insensitive abi1 and abi2 mutants. The Dc3 promoter directed ABA- and mannitol-inducible GUS expression in Arabidopsis guard cells and the two treatments were additive. The aba1, abi1, and abi2 mutant genotypes had reduced GUS expression in guard cells of cotyledons in response to mannitol, whereas abi1 and abi2 mutants were reduced in ABA-inducible GUS expression, consistent with overlapping ABA- and drought-response pathways. Quantitative fluorometric GUS assays of leaf extracts showed that abi2 mutants responded less to exogenous ABA than did abi1 mutants, and abi2 mutants responded more to mannitol than did abi1 mutants. We conclude that Dc3-GUSArabidopsis is a tractable system in which to study tissue-specific ABA and drought signalling and suggest that ABI2 functions predominantly over ABI1 in COR78 and COR47 gene expression and guard-cell Dc3-GUS expression. Received: 23 May 1999 / Accepted: 3 December 1999     

The seed coat-specific expression of a subtilisin-like gene, SCS1, from soybean

A seed coat-specific gene, SCS1 (Seed Coat Subtilisin 1), from soybean, Glycine max [L.] Merill, has been identified and studied. The gene belongs to a small family of genes with sequence similarity to the subtilisins, which are serine proteases. Northern blot analysis showed that SCS1 RNA accumulates to maximal levels in seed coats at 12 days post anthesis, preceding the final stages of seed coat differentiation. The SCS1 RNA was not found in other tissues including embryos, seed pods, flowers, stems, roots or leaves. In-situ hybridization studies confirmed the temporal pattern of expression observed by Northern blot analysis and further revealed a restricted pattern of RNA accumulation in thick-walled parenchyma cells of the seed coats. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. The tissue-specific subtilisin-like gene may be required for regulating the differentiation of the thick-walled parenchyma cells. Received: 10 January 2000 / Accepted: 22 February 2000     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 2000     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

Three maize root-specific genes are not correctly expressed in regenerated caps in the absence of the quiescent center

The quiescent center is viewed as an architectural template in the root apical meristem of all angiosperm and gymnosperm root tips. In roots of Arabidopsis thaliana (L.) Heynh., the quiescent center inhibits differentiation of contacting initial cells and maintains the surrounding initial cells as stem cells. Here, the role of the quiescent center in the development of the maize (Zea mays L.) root cap has been further explored. Three maize root-specific genes were identified. Two of these were exclusively expressed in the root cap and one of them encoded a GDP-mannose-4,6-dehydratase. Most likely these two genes are structural, tissue-specific markers of the cap. The third gene, a putative glycine-rich cell wall protein, was expressed in the cap and in the root epidermis and, conceivably is a positional marker of the cap. Microsurgical and molecular data indicate that the quiescent center and cap initials may regulate the positional and structural expression of these genes in the cap and thereby control root cap development. Received: 22 September 1999 / Accepted: 9 November 1999     

Hormonal regulation of fruitlet abscission induced by carbohydrate shortage in citrus

 The hormonal signals controlling fruitlet abscission induced by sugar shortage in citrus were identified in Satsuma mandarin, Citrus unshiu (Mak.) Marc, cv. Clausellina and cv. Okitsu. Sugar supply, hormonal responses and fruitlet abscission were manipulated through full, partial or selective leaf removals at anthesis and thereafter. In developing fruitlets, defoliations reduced soluble sugars (up to 98%), but did not induce nitrogen and water deficiencies. Defoliation-induced abscission was preceded by rises (up to 20-fold) in the levels of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in fruitlets. Applications to defoliated plants showed that ABA increased ACC levels (2-fold) and accelerated fruitlet abscission, whereas norflurazon and 2-aminoethoxyvinyl glycine reduced ACC (up to 65%) and fruitlet abscission (up to 40%). Only the full defoliation treatment reduced endogenous gibberellin A₁ (4-fold), whereas exogenous gibberellins had no effect on abscission. The data indicate that fruitlet abscission induced by carbon shortage in citrus is regulated by ABA and ACC originating in the fruits, while gibberellins are apparently implicated in the maintenance of growth. In this system, ABA may act as a sensor of the intensity of the nutrient shortage that modulates the levels of ACC and ethylene, the activator of abscission. This proposal identifies ABA and ACC as components of the self-regulatory mechanism that adjusts fruit load to carbon supply, and offers a physiological basis for the photoassimilate competition-induced abscission occurring under natural conditions. Received: 19 February 1999 / Accepted: 14 August 1999     

Gravity independence of seed-to-seed cycling in Brassica rapa

 Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity. Received: 3 August 1999 / Accepted: 27 August 1999     

Micromechanics and anatomical changes during early ontogeny of two lianescent Aristolochia species

 The mechanical properties of young stems of Aristolochia macrophylla Lam. and Aristolochia brasiliensis Mart. et Zucc. were studied during elongation growth and primary differentiation. Data for the modulus of elasticity, for the viscoelastic behaviour caused by longitudinal tension and for the shear modulus resulting from torsion around a longitudinal axis were related to the underlying structural changes by quantitative analysis of stem anatomy, tissue distribution, ultrastructure, and cell wall biochemistry. The orientation of cellulose microfibrils was determined by light microscopy and small-angle X-ray diffraction, and the lignin content was determined by thioglycolic acid derivatization and spectroscopic quantification. It was demonstrated that the increase in stability during early development is due to the complementary effects of increase in cell wall material, lignification, and cellulose microfibril alignment. A detailed micromechanical model, considering internal prestresses, is proposed to explain the characteristic biphasic stress-strain behaviour as well as the strain-hardening observed. Received: 22 March 1999 / Accepted 9 September 1999     

Analytical electron microscopical investigations on the apoplastic pathways of lanthanum transport in barley roots

 In transmission electron microscopy studies, lanthanum ions have been used as electron-opaque tracers to delineate the apoplastic pathways for ion transport in barley (Hordeum vulgare L.) roots. To localize La³⁺ on the subcellular level, e.g. in cell walls and on the surface of membranes, electron-energy-loss spectroscopy and electron-spectroscopic imaging were used. Seminal and nodal roots were exposed for 30 min to 1 mM LaCl₃ and 10 mM LaCl₃, respectively. In seminal roots, possessing no exodermis, La³⁺ diffusion through the apoplast was stopped by the Casparian bands of the endodermis. In nodal roots with an exodermis, however, La³⁺ diffusion through the cortical apoplast had already stopped at the tight junctions of the exodermal cell walls resembling the Casparian bands of the endodermis. Therefore, we conclude that in some specialized roots such as the nodal roots of barley, the physiological role of the endodermis is largely performed by the exodermis. Received: 28 July 1999 / Accepted: 24 February 2000